RE: [Histonet] MSH6

From:

"Barry Madigan"

Hello Josie,
The antibodies for mismatch repair genes that we use consists of MLH1, MSH2
and MSH6 all of which we obtain from BD (Becton Dickinson).
They are all performed on our BOND MAX Immunostainer using the high pH
retrieval solution with EDTA (30 min) and a Vision Polymer Detection System.
We have no troubles with them except sometimes we find that with archival
cases we need to apply extra heat for MLH1.
When this extra step is performed the MLH1 internal controls come up
beautifully.
Before we had the BOND MAX we used to heat retrieve using a microwave
pressure cooker for 8 minutes on high in TRIS/EDTA pH 9.0 for MLH1.
For the MSH6 antibody we currently obtain a dilution of 1:200.
MSH2 1:500 and MLH1 1:100.
Hope this is of some help.
Regards
Barry
Barry Madigan
Immunohistochemistry
QHPS-Royal Brisbane Hospital Campus
Australia
-----Original Message-----
From: histonet-bounces@lists.utsouthwestern.edu
[mailto:histonet-bounces@lists.utsouthwestern.edu] On Behalf Of Orr, Rebecca
Sent: Saturday, 4 February 2006 1:24 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] MSH6
Hi Josie,
How many different types of protocols did you try on the XT?
With the different temp and Cell conditioning options I'm surprised you
didn't get something.
Are you running the MLH-1 and MSH-2 as well? How are they?
My experience with the Microsateliite Instabilities is 4+ staining in 2
out of 3 antibodies, depending on the control tissue. The third
antibody would compare at 2+ or a bit stronger.
I have the best of both worlds with a Benchmark and a Nemesis. I
experienced the poor staining results of these particular antibodies and
I decided to run them on the Nemesis with Biocare polymer detection and
they are gorgeous. I use the Biocare antibodies, too.
In case you don't have that option, I would consider trying another
control tissue, and running through all the CC options. I'm not sure
if they behave differently without heat, did you try running a protocol
with out heat in the detection?
Do you have access to the polymer from Ventana? Maybe try that? Or run
the antibody at 1:5 or 1:10.
Or do you have a pressure cooker to try the HEIR offline. If you get
good staining with offline HEIR then you can rule out the detection as
the issue, too.
Becky
Becky Orr CLA,HT(ASCP)
IHC Lead
Evanston Northwestern Healthcare
847-570-2771
-----Original Message-----
Message: 8
Date: Wed, 1 Feb 2006 13:54:36 -0600
From: "Nava, Josefa"
Subject: [Histonet] looking for Antibodies-MSH6 and PMS2
To:
Message-ID:
<2C515C1049EAF5459EFD8C9B929078A41945A8@phdex03.txhealth.org>
Content-Type: text/plain; charset="us-ascii"
Hello Everyone ,
I am using Ventana BMK/XT , can someone tell me a good source of MSH6
and PMS2 that will work on the Ventana Machine. I appreciate any
information. Thank you.
Josie
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